Datura species are valued ornamentals but contain toxic tropane alkaloids (TAs) like hyoscyamine and scopolamine, which restrict their safe horticultural use. To address this, we developed a genome editing platform in Datura inoxia for creating non-toxic varieties. We first established an efficient, auxin-independent shoot regeneration system using a novel cytokinin combination (thidiazuron and 6-benzylaminopurine) achieving over 7 shoots per explant. This system facilitated an Agrobacterium tumefaciens-mediated transformation protocol with a stable efficiency exceeding 50% (49 independent lines from 100 explants for LS; 36 lines from 70 explants for CYP80F1). Using this platform, we performed CRISPR/Cas9-mediated knockout of two key TA biosynthetic genes, LS (littorine synthase) and CYP80F1 (littorine mutase). Among the transgenic lines analyzed, 8 out of 15 (53%) carried mutations in LS, while all 12 (100%) lines carried mutations in CYP80F1. HPLC and high-resolution mass spectrometry confirmed the complete absence of hyoscyamine and scopolamine in the mutant leaves, with no detectable peaks at the corresponding retention times. Crucially, the edited plants grew normally and were morphologically indistinguishable from the wild type. This work establishes the first CRISPR/Cas9 platform for Datura and generates the first non-toxic germplasm, providing both a functional genomics tool and a foundation for breeding safe ornamental cultivars.
Zou et al. (Sun,) studied this question.